Understanding Downstream Processing in Bioprocessing

 
 
Chapter 8
Downstream processing
 
 
Outline
 
Introduction
Cell disruption
Removal of insoluble
Product isolation
Product purification
Product polishing
 
 
 Stock Culture
Shake Flasks
Raw Materials
Sterilization
 Air
Agitator
Recovery
Purification
Products
Seed fermenter
Medium Formulation
 
Typical Bioprocessing
 
Downstream processing
 
The various stages of processing that occur after the
completion of the fermentation or bioconversion
stage, including 
separation
, 
purification
, and
packaging
 of the product.
 
is any treatment of culture broth after fermentation to
concentrate
 and 
purify
 the products
 
Stage in Downstream processing
Crude product
Cell disruption
Removal of insoluble
Product recovery
Purify the product
Finished product
market
 
   Cell disruption
 
Some product are intercellular including many
enzymes and products required to disrupt the cells
and release these products e.g. Yeast
Cell disruption can be achieved by both mechanical
and non mechanical methods
Mechanical – sonication and liquid shear
homogenization etc.
Non mechanical – autolysis, osmosis shock etc.
 
Removal of Insoluble
 
Separation of cells, cell debris or other particulate
matter from fermentation broth culture
Typical operations to achieve this:
1)
Filtration
2)
Centrifugation
3)
Sedimentation
4)
Flocculation a process where a solute comes out
of solution in the form of floc or flakes.
5)
Gravity settling
 
Filtration
 
A mechanical operation used for the separation
of solids from fluids (liquids or gases) by
interposing
 a medium to 
porous membrane
through which the fluid can 
pass
, but the solids
in the fluid are 
retained
.
 
                  
Filtration
 
Separation of particles from liquid by applying a
pressure to the solution to force the solution through
a filter. Filter are materials with pores
Particles 
larger
 than the 
pore size 
of the filter are
retained
 by filter.
Particles 
smaller
 than the 
pore size 
of the filter 
pass
through the filter along with the liquid
 
Filtration
 
The solid particles deposited on the filter form
a layer, which is known as 
filter cake
.
All the solid particles from the feed are
stopped by the cake ,and the cake grows at the
rate at which particles are bought to its surface.
All of the fluid goes through the 
cake 
and 
filter
medium.
 
 
Continuous Rotary Vacuum filter
 
It is one of the most commonly used type of
filter in fermentation.
The drum is pre coated prior to filtration.
A small agent of coagulating is added to the
broth before  it is pumped into the filter.
The drum rotates under vacuum and a thin
layer of cells sticks to the drum.
The thickness of the layer increases in the
section designed for forming the cake.
 
Liquid filtrate
 
Cell mass
 
Continuous Rotary filter
 
Knife
 
Hollow spokes
 
Filter
 
Perforate
d drum
 
Points to be considered while
selecting the filter medium:
 
Ability to build the solid.
Minimum resistance to flow the filtrate.
Resistance to chemical attack.
Minimum cost.
Long life
 
Centrifugation
 
Centrifugation is used to separate particles of 100 – 0.1
micrometer from liquid by 
gravitational forces
.
It depends on particles size, density difference between
the cells and the broth and broth viscosity.
Use of the centrifugal force for the separation of
mixtures
More-dense 
components migrate 
away
 from the axis of
the centrifuge
Less-dense
 components  migrate 
towards
 the axis.
Types of centrifuges used are Tubular bowl centrifuge,
multi-chamber  centrifuge, disc bowl centrifuge etc.
 
Sedimentation
 
It is applicable only for large particles greater
than 100 micrometer flocs.
It is a slow process and takes ~3 hours.
It is used in process like activated sludge
effluent treatment.
It’s a free settling process depends only on
gravity.
 
Flocculation
 
Process where a solute comes out of solution in
the form of 
flocs
 or 
flakes
.
Particles finer than 0.1 µm in water remain
continuously in motion due to electrostatic charge
which causes them to repel each other
Once their electrostatic charge is neutralized (use
of coagulant) the finer particles start to collide
and combine together .
These larger and heavier particles are called flocs.
 
Product Isolation
 
Removal of those components whose properties
vary markedly from that of the 
desired product
.
Water is the chief impurity
a)
Isolation steps are designed to remove it
(i.e.dialysis)
b)
Reducing the volume
c)
Concentrating the product.
d)
Liquid –liquid extraction, adsorption,
ultrafiltration, and precipitation are some of the
unit operations involved
.
 
Liquid -Liquid extraction
 
It is a separation process that takes the advantage of the
relative solubilities of solute in immiscible solvents.
Solute is dissolved more readily and becomes more
concentrated in the solvent in which it has a higher
solubility.
A partial separation occurs when a number of solutes
have different relative solubilities  in the two solvents
used.
Solvent should be non toxic, selective, inexpensive and
immiscible with broth and should have a high
distribution coefficient for the product.
 
Adsorption
 
is a surface phenomenon
It is the binding of molecules to the 
surface
 and
different from absorption.
The binding to the surface is weak and reversible.
Compounds containing chromogenic group are
usually strongly adsorbed on activated carbon.
Common adsorbent used are activated carbon,
silica gel ,alumina becoz they present enormous
surface areas per unit weight
.
 
Ultrafiltration
 
UF is basically a pressure-driven separation
process.
The operating pressure is usually between 0.1
and 1 MPa.
 
Ultrafiltration
 
UF is governed by a screening principle and
dependent on particle size.
UF membranes have a pore size between 1 nm
and 100 nm (10 and 2000 Å), thus allowing
retention of compounds with a molecular
weight of 300 to 500 000 Dalton.
Typically, the process is suitable for retaining
biomolecules, bacteria, viruses, polymers,
colloidal particles and sugar molecules.
 
Ultrafiltration
 
Precipitation
 
Formation of a solid in a solution during a
chemical reaction.
Solid formed is called the 
precipitate 
and the
liquid remaining above the solid is called the
supernate.
 
Precipitation
 
Salts such as ammonium & sodium sulphate
are used for proteins to precipitate.
Organic solvents methanol used to precipitate
dextrans.
Chilled ethanol and acetone used for protein
precipitation.
Non ionic polymer such as polyethylene glycol
used in precipitation.
 
Product Purification
 
Done to separate those contaminants that
resemble
 the product 
very closely 
in physical and
chemical properties.
Expensive to carry out
Require sensitive and sophisticated equipment
Significant fraction of the entire downstream
processing expenditure.
Examples of operations include affinity, size
exclusion, reversed phase chromatography,
crystallization and fractional precipitation
.
 
Product purification
 
Removing components with properties similar to
those of the products
 
Chromatography
 
Separates molecules by their  chemical and physical
difference
 
e.g
.
 
Adsorption chromatography
Affinity chromatography
Ion exchange chromatography
 
Components
 
- Solute: molecule of interest
 
- Mobile phase( solvent, gas)MF
 
- Stationary phase( solid, liquid)SF
 
Principle
 
- The solute partion between the two phase
 
- The interaction with the solute phase determines how
fast the solute migrate in the mobile phase
 
- Solute that interact differently with the stationary
phase can be separated
 
Ch
r
omatography
 
components
 
and princ
i
ple
 
Liquid Chromatography
 
Mobile phase is a liquid.
Carried out either in a column or a plane.
HPLC
In the HPLC technique, the sample is forced
through a column that is packed with
irregularly or spherically shaped particles or a
porous monolithic layer (stationary phase) by
a liquid (mobile phase) at high pressure.
 
HPLC Configuration
 
Product Polishing
 
Final processing steps  which end with
packaging of the product in a form that is
stable and easily transportable
 
Crystallization, concentration and drying
are typical unit operations
 
Crystallization
 
process of formation of solid crystals
precipitating from a solution, melt or more
rarely deposited directly from a gas.
 
chemical solid-liquid separation technique, in
which 
mass transfer 
of a 
solute
 from the 
liquid
solution
 to a 
pure solid crystalline 
phase
occurs.
 
Crystallizat
i
on
 
p
r
ocess
 
sol
i
d
 
pha
s
e
 
 
 
Ge
n
e
r
a
t
i
on
 
of
s
u
p
e
r
s
a
tu
r
a
t
i
on:
dr
i
ving
 
f
o
r
c
e
 
 
L
i
quid
 
m
i
x
er
 
 
 
Nuc
l
e
a
t
i
on:
 
bi
r
th
 
of
 
 
 
 
 
 
 
Final
 
p
r
oduct
 
C
r
y
s
t
al g
r
o
w
t
h
 
 
Cryst
a
llization
 
 
B
y
 
c
ool
i
ng
 
s
a
tu
r
a
t
ed
 
sol
u
tion
 
Drying
 
 
It i
n
v
ol
v
es
 
the
 
t
r
an
s
f
er
 
of
 
he
a
t
 
t
o
 
the
 
w
e
t
 
m
a
t
er
i
al
 
and
 
the
 
r
em
o
v
al
 
of
 
the
 
moi
s
tu
r
e
 
as
 
wa
t
er
 
v
apor
 
 
Dry
i
ng
 
M
e
thods
1
 
A
tmosp
h
er
i
c
 
dry
i
ng
 
2
V
acuum
 
dry
i
ng
 
(De
c
omp
r
ess
i
on
 
)
 
3
 
F
r
e
e
z
e
-
dry
i
ng
 
(l
y
oph
i
l
i
z
a
tio
n
)
4
 
Sp
r
a
y
 
dry
i
ng
 
lyophil
i
zation
 
Freeze drying
Process in which water is removed from a product
after it is frozen.
It is placed under vacuum allowing the ice to
change directly from the solid to vapor without
passing through a liquid phase
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Downstream processing plays a crucial role in bioprocessing after fermentation, focusing on separating, purifying, and packaging the final product. It involves stages like cell disruption, removal of insoluble particles, product recovery, and purification methods such as filtration and centrifugation. Various techniques like mechanical and non-mechanical processes are used for cell disruption to release intracellular products. The overall goal is to obtain a highly purified end product ready for the market.


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  1. Chapter 8 Downstream processing

  2. Outline Introduction Cell disruption Removal of insoluble Product isolation Product purification Product polishing

  3. Typical Bioprocessing Stock Culture Raw Materials Medium Formulation Shake Flasks Sterilization Seed fermenter Air Agitator Recovery Purification Products

  4. Downstream processing The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product. is any treatment of culture broth after fermentation to concentrate and purify the products

  5. Stage in Downstream processing Crude product Product recovery Removal of insoluble Cell disruption Purify the product Finished product market

  6. Cell disruption Some product are intercellular including many enzymes and products required to disrupt the cells and release these products e.g. Yeast Cell disruption can be achieved by both mechanical and non mechanical methods Mechanical sonication and liquid shear homogenization etc. Non mechanical autolysis, osmosis shock etc.

  7. Removal of Insoluble Separation of cells, cell debris or other particulate matter from fermentation broth culture Typical operations to achieve this: 1) Filtration 2) Centrifugation 3) Sedimentation 4) Flocculation a process where a solute comes out of solution in the form of floc or flakes. 5) Gravity settling

  8. Filtration A mechanical operation used for the separation of solids from fluids (liquids or gases) by interposing a medium to porous membrane through which the fluid can pass, but the solids in the fluid are retained.

  9. Filtration Separation of particles from liquid by applying a pressure to the solution to force the solution through a filter. Filter are materials with pores Particles larger than the pore size of the filter are retained by filter. Particles smaller than the pore size of the filter pass through the filter along with the liquid

  10. Filtration The solid particles deposited on the filter form a layer, which is known as filter cake. All the solid particles from the feed are stopped by the cake ,and the cake grows at the rate at which particles are bought to its surface. All of the fluid goes through the cake and filter medium.

  11. Continuous Rotary Vacuum filter It is one of the most commonly used type of filter in fermentation. The drum is pre coated prior to filtration. A small agent of coagulating is added to the broth before it is pumped into the filter. The drum rotates under vacuum and a thin layer of cells sticks to the drum. The thickness of the layer increases in the section designed for forming the cake.

  12. Continuous Rotary filter Liquid filtrate Filter Hollow spokes Cell mass Perforate d drum Knife

  13. Points to be considered while selecting the filter medium: Ability to build the solid. Minimum resistance to flow the filtrate. Resistance to chemical attack. Minimum cost. Long life

  14. Centrifugation Centrifugation is used to separate particles of 100 0.1 micrometer from liquid by gravitational forces. It depends on particles size, density difference between the cells and the broth and broth viscosity. Use of the centrifugal force for the separation of mixtures More-dense components migrate away from the axis of the centrifuge Less-dense components migrate towards the axis. Types of centrifuges used are Tubular bowl centrifuge, multi-chamber centrifuge, disc bowl centrifuge etc.

  15. Sedimentation It is applicable only for large particles greater than 100 micrometer flocs. It is a slow process and takes ~3 hours. It is used in process like activated sludge effluent treatment. It s a free settling process depends only on gravity.

  16. Flocculation Process where a solute comes out of solution in the form of flocs or flakes. Particles finer than 0.1 m in water remain continuously in motion due to electrostatic charge which causes them to repel each other Once their electrostatic charge is neutralized (use of coagulant) the finer particles start to collide and combine together . These larger and heavier particles are called flocs.

  17. Product Isolation Removal of those components whose properties vary markedly from that of the desired product. Water is the chief impurity a) Isolation steps are designed to remove it (i.e.dialysis) b) Reducing the volume c) Concentrating the product. d) Liquid liquid extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.

  18. Liquid -Liquid extraction It is a separation process that takes the advantage of the relative solubilities of solute in immiscible solvents. Solute is dissolved more readily and becomes more concentrated in the solvent in which it has a higher solubility. A partial separation occurs when a number of solutes have different relative solubilities in the two solvents used. Solvent should be non toxic, selective, inexpensive and immiscible with broth and should have a high distribution coefficient for the product.

  19. Adsorption is a surface phenomenon It is the binding of molecules to the surface and different from absorption. The binding to the surface is weak and reversible. Compounds containing chromogenic group are usually strongly adsorbed on activated carbon. Common adsorbent used are activated carbon, silica gel ,alumina becoz they present enormous surface areas per unit weight.

  20. Ultrafiltration UF is basically a pressure-driven separation process. The operating pressure is usually between 0.1 and 1 MPa.

  21. Ultrafiltration UF is governed by a screening principle and dependent on particle size. UF membranes have a pore size between 1 nm and 100 nm (10 and 2000 ), thus allowing retention of compounds with a molecular weight of 300 to 500 000 Dalton. Typically, the process is suitable for retaining biomolecules, bacteria, viruses, polymers, colloidal particles and sugar molecules.

  22. Ultrafiltration

  23. Precipitation Formation of a solid in a solution during a chemical reaction. Solid formed is called the precipitate and the liquid remaining above the solid is called the supernate.

  24. Precipitation Salts such as ammonium & sodium sulphate are used for proteins to precipitate. Organic solvents methanol used to precipitate dextrans. Chilled ethanol and acetone used for protein precipitation. Non ionic polymer such as polyethylene glycol used in precipitation.

  25. Product Purification Done to separate those contaminants that resemble the product very closely in physical and chemical properties. Expensive to carry out Require sensitive and sophisticated equipment Significant fraction of the entire downstream processing expenditure. Examples of operations include affinity, size exclusion, reversed phase chromatography, crystallization and fractional precipitation.

  26. Product purification Removing components with properties similar to those of the products Chromatography Separates molecules by their chemical and physical difference Adsorption chromatography Affinity chromatography Ion exchange chromatography e.g.

  27. Components - Solute: molecule of interest - Mobile phase( solvent, gas)MF - Stationary phase( solid, liquid)SF Principle - The solute partion between the two phase - The interaction with the solute phase determines how fast the solute migrate in the mobile phase - Solute that interact differently with the stationary phase can be separated

  28. Chromatography components and principle

  29. Liquid Chromatography Mobile phase is a liquid. Carried out either in a column or a plane. HPLC In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure.

  30. HPLC Configuration

  31. Product Polishing Final processing steps which end with packaging of the product in a form that is stable and easily transportable Crystallization, concentration and drying are typical unit operations

  32. Crystallization process of formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas. chemical solid-liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.

  33. Crystallization process Generation of supersaturation: driving force Nucleation: birth of Liquid mixer solid phase Final product Crystal growth

  34. Crystallization By cooling saturated solution

  35. Drying It involves the transfer of heat to the wet material and the removal of the moisture as water vapor Drying Methods 1 Atmospheric drying 2 Vacuum drying (Decompression ) 3 Freeze-drying(lyophilization) 4 Spray drying

  36. lyophilization Freeze drying Process in which water is removed from a product after it is frozen. It is placed under vacuum allowing the ice to change directly from the solid to vapor without passing through a liquid phase

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